Lysis buffer

Western blotting was performed to corroborate the IHC results. BT20, MDA-MB-231, or HUVEC cells were lysed using lysis buffer (Amresco, Solon, OH, USA) supplemented with 0.1% protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was determined using a DC assay (Bio-Rad, Hercules, CA, USA) per manufacturer instructions. 15 μg of protein from each cell line was loaded into 8% Bis-Tris polyacrylamide gels (LifeTech, Carlsbad, CA, USA), and gels were run in 1X MES-SDS (2-(N-morpholino)ethanesulfonic acid-sodium dodecyl sulfate) running buffer for 35 min at 165 V. Protein was transferred onto nitrocellulose membranes for 15 min using a 1-step transfer system (Thermo Fisher Scientific). Membranes were blocked in 5% PBSA with 0.1% Tween-20 (PBST) for 1 hr and then incubated with 4 μg/mL goat-anti-mouse EGFR antibody (Santa Cruz Biotechnology) in 5% PBSA overnight at 4°C. After overnight incubation, membranes were washed 3 times with PBST and then incubated with 0.24 μg/mL HRP-AM (Santa Cruz Biotechnology) for 30 min at room temperature. Lastly, membranes were rinsed 3 times with PBST, reacted with VisiGlo electrochemiluminescent reagent (VWR) for 5 min, and imaged using the ChemiDoc-iT™2 Imager (UVP, Upland, CA, USA).